JP2010535800A - Nucleic acid-lipopolymer composition - Google Patents

Abstract

  Compositions, methods and applications are provided that enhance the efficiency of nucleic acid transfection. In one embodiment, the pharmaceutical composition can comprise a concentration of at least about 0.5 mg / ml nucleic acid condensed with a cationic lipopolymer suspended in an isotonic solution, wherein the cationic lipopolymer comprises cholesterol. And a polyethylene polymer glycol covalently linked cationic polymer backbone, wherein the molar ratio of cholesterol to cationic polymer backbone is in the range of about 0.1 to about 10, and the polyethylene glycol to cationic polymer backbone mole The ratio is in the range of about 0.1 to about 10. The composition can further comprise a filler excipient.

Description

  The present invention relates to concentrated and stable formulations comprising nucleic acids and lipopolymers, their compositions, preparation methods and applications. The present invention thus relates to the fields of molecular biology and biochemistry.

  Synthetic gene delivery vectors have significant advantages over viral vectors due to better safety compliance, simple chemistry and cost-effective manufacturing. However, because synthetic vectors have lower transfection efficiency compared to viral vectors, most developments in synthetic gene delivery systems have focused on improving delivery efficiency. As a result, little attention has been directed to the pharmaceutical development of synthetic delivery systems, despite problems found in formulation stability, scale-up and dosing flexibility. Drugs containing DNA that self-assemble to form nanoparticles often exhibit low stability, especially when the formulation is an aqueous suspension. In such formulations, DNA with synthetic vectors generally aggregates over time, especially at concentrations required for optimal dosing in the clinical setting. Such formulations are often difficult to prepare at DNA concentrations> 0.3 mg / ml, with limited commercial applications, especially in topical delivery where flexible dosing is limited due to volume constraints End up. DNA aggregation reduces or eliminates DNA activity, making the composition unsuitable for therapeutic use.

  This physical instability is one of the reasons underlying the loss of transfection activity. Particle rupture or expression of fusion due to high bending of the lipid bilayer or physical dissociation of the lipid from DNA is also a potential basis for the low stability and aggregation of cationic lipid-based gene delivery complexes The reason is assumed. Chemical modifications such as oxidative hydrolysis of delivery vectors may also contribute to particle instability.

  Due to its low stability, DNA preparations were required to be prepared clinically in early clinical trials. The inability to prepare and store clinical products at the concentrations required for optimal dosing has become a major obstacle to wide clinical practice and commercialization of non-viral DNA products. This necessitates training of doctors for prescription of medicines and imposes on-site quality control measures.

  Freeze drying is a useful method to improve the long-term stability of some pharmaceuticals. However, this process tends to change the physicochemical properties of the drug product and leads to aggregation and loss of transfection upon reconstitution, and is therefore generally not suitable for drying DNA complexes with synthetic vectors.

  Several approaches have been attempted to prevent aggregation and damage of the formulation during lyophilization. In certain cases, lyophilization of DNA complexes in the presence of cryoprotectants such as low molecular weight sugars, dextran and polyethylene glycol can also provide better stability to the product, This approach does not appear to improve medication flexibility. The addition of sugar is often the most commonly used approach for this purpose. Many of the sugars tested have been found to prevent, to some extent, formulation damage and particle aggregation, but the quality of this action varies with the type of sugar and the delivery vector used.

  Although lyophilization provides some improvement in the shelf life of the formulation, the conditions necessary to produce a lyophilized DNA product allow only limited pharmaceutical applications. Even with the most effective cryoprotective sugars, very high sugar / DNA molar ratios (generally over 1000: 1) are required for stability. As a result, the lyophilized product often has to be diluted at a very high magnification in order to obtain an isotonic formulation, and the final lyophilized DNA concentrate A decrease in DNA concentrate occurs. For many cationic carriers, the final DNA concentration can generally be about 0.1-0.2 mg / ml, often below 0.1 mg / ml. Low-concentration formulations are sufficient for in vitro studies but their clinical application may be limited due to the large volume required for optimal dosing. For example, at the optimal sugar concentration required for stability, a 1 mg dose of DNA may need to be diluted with 5-10 ml in order to maintain isotonicity. Too much volume for typical in vivo administration. This formulation limitation hinders flexible dosing and is one of the major causes of suboptimal efficacy of synthetic gene delivery systems in human clinical trials, which is stable and biologically active, This is the basis for the need for higher concentrations of DNA preparations.

  The present invention provides compositions that exhibit unexpected stability at high nucleic acid concentrations and improve the efficacy and dosage flexibility of nucleic acid transfection. The compositions described herein can be efficiently lyophilized and reconstituted to various nucleic acid concentrations, including high nucleic acid concentrations, without loss of nucleic acid biological activity or aggregation.

  In one aspect, the present invention provides a composition, preferably a pharmaceutical composition, comprising a mixture of a cationic lipopolymer and at least about 0.5 mg / ml nucleic acid, wherein the mixture is suspended in an aqueous solution. The cationic lipopolymer has a cationic polymer backbone in which cholesterol and polyethylene glycol groups are independently covalently bonded. The molar ratio of cholesterol to cationic polymer backbone is in the range of about 0.1 to about 10, and the molar ratio of polyethylene glycol to cationic polymer backbone is in the range of about 0.1 to about 10. The composition may further comprise a filler excipient. In certain embodiments, the mixture of nucleic acid and lipopolymer forms a complex. In certain embodiments, the composition comprises a condensed nucleic acid. The amount of nucleic acid to be condensed generally depends on the compositional structure of the nucleic acid and the conditions under which the composition is prepared.

  The present invention further provides a method of making the aforementioned composition.

  In another aspect, the present invention provides a lyophilized composition of nucleic acid and lipopolymer. The lyophilized composition of the present invention, preferably the lyophilized pharmaceutical composition, comprises a mixture of filler excipient, condensed nucleic acid and cationic lipopolymer. As mentioned above, the cationic lipopolymer is covalently linked with cholesterol and polyethylene glycol, and the molar ratio of cholesterol to cationic polymer backbone is in the range of about 0.1 to about 10, and the polyethylene glycol to cationic polymer backbone is Having a cationic polymer backbone with a molar ratio in the range of about 0.1 to about 10.

  The present invention further provides methods for using the compositions described herein in the treatment of diseases and / or disorders, eg, by transfecting various cells and tissues.

It is the schematic which shows the manufacturing process of this invention. It is a graph which shows the particle size of the nucleic acid in a concentration state and a non-concentration state. It is a figure which shows the result of an electrophoresis experiment in order to show condensation of a nucleic acid. Figure 6 is a graph showing transfection activity according to a further embodiment of the invention. It is the photograph of the nerve slice which shows the treatment result by the lipopolymer which does not use IL-12. It is the photograph of the nerve slice which shows the treatment result by a lipopolymer using IL-12. 2 is two graphs showing the anticancer effect of IL-12 by lipopolymer compared to a control. 2 is a graph showing the particle size of various nucleic acid / cationic polymer mixtures. 2 is a graph showing the particle size of various nucleic acid / cationic polymer mixtures. Figure 2 is a graph showing luciferase expression resulting from various nucleic acid / cationic polymer mixtures. Figure 2 is a graph showing luciferase expression resulting from various nucleic acid / cationic polymer mixtures. 2 is a graph showing the biological activity of a nucleic acid / cationic lipopolymer composition after long-term storage.

  Before the present invention is disclosed and described, the present invention is not limited to the specific structures, process steps, or materials disclosed herein, but is recognized by those skilled in the relevant art. It should be understood that it extends to the equivalent of. Moreover, the terminology used herein is for the purpose of describing particular embodiments only, since the scope of the present invention is limited only by the appended claims and equivalents thereof. It should also be understood that there is no intent to limit the scope.

  As used in this specification and the appended claims, the singular expressions “a”, “a”, “a”, “an”, and “this” (the) clearly state otherwise. It should be noted that plurals are included unless otherwise noted.

  As used herein, the terms “condensed nucleic acid” and “partially condensed nucleic acid” are used to refer to nucleic acids contacted by a cationic lipopolymer of the present invention. In certain embodiments, the condensed nucleic acid remains in contact with the cationic lipopolymer. Condensed nucleic acids generally occupy significantly less volume than non-condensed nucleic acids. However, it is recognized that the amount of condensed nucleic acid can vary depending on the local environment (eg, lipids resist an aqueous environment, etc.). In various aspects of the invention, the condensed nucleic acid is a nanoparticle of nucleic acids and cationic lipopolymers having a size of about 50 nm to about 300 nm, more preferably about 50-200, even more preferably about 50-150 nm. It is in particle form. A “partially condensed nucleic acid” is a nucleic acid that has been contacted by the cationic lipopolymer of the present invention, in which the nucleic acid has not yet fully condensed, but is still not condensed. Refers to those that occupy significantly less volume.

  The term “complex” as used herein means a nucleic acid that is bound to a lipopolymer, preferably a cationic lipopolymer. The complex comprising the condensed nucleic acid and the cationic lipopolymer is usually present as particles, preferably nanoparticles.

  As used herein, the terms “transfect” and “transfection” refer to the transfer of nucleic acids from the environment external to the cell to the cell's internal environment, particularly in relation to the cytoplasm and / or cell nucleus. . Without being bound by any particular theory, the nucleic acid is encapsulated in, adhering to, or entrained in one or more cationic polymer / nucleic acid complexes and then into the cell. It should be understood that it can be delivered. Certain transfection instances deliver nucleic acids to the cell nucleus.

  As used herein, a “subject” refers to a mammal that can benefit from the administration or method of a pharmaceutical composition of the invention. Examples of subjects include humans and may also include animals such as horses, pigs, cows, dogs, cats, rabbits, and aquatic mammals.

  As used herein, “composition” refers to a mixture of two or more compounds, elements or molecules. In some embodiments, the term “composition” may be used to refer to a mixture of a nucleic acid and a delivery system.

  As used herein, “N: P ratio” refers to the molar ratio of amine nitrogen in the functional cationic lipopolymer and phosphate groups in the nucleic acid.

  As used herein, “physicochemical properties” include, but are not limited to, various particle sizes and surface charges of nucleic acid complexes with cationic polymers, pH and osmolality of particle solutions, etc. Refers to characteristics.

  As used herein, the terms “administering”, “administering” and “delivering” refer to a method of providing a composition to a subject. Administration can be accomplished by various routes known in the art such as oral, parenteral, transdermal, inhalation, implantation. Thus, oral administration can be accomplished by swallowing, chewing, aspiration of an oral dosage form containing the composition. Parenteral administration can be accomplished by injecting the composition intravenously, intraarterially, intramuscularly, intraarticularly, intrathecally, intraperitoneally, subcutaneously, and the like. Injectables for such applications are in conventional form, either as liquid solutions or suspensions, or in solid form suitable for preparation as solutions or suspensions in liquid prior to injection. Or as an emulsion. Further, transdermal administration can be achieved by applying, pasting, rolling, adhering, pouring, pressing, rubbing, etc. the transdermal composition to the skin surface. These and additional methods of administration are well known in the art. In one particular embodiment, administration also includes delivering the composition to a subject so that it binds to the target cells so that the composition circulates systemically and is taken up by endocytosis. it can.

  As used herein, the term “nucleic acid” also refers to DNA and RNA and their synthetic congeners. Examples of nucleic acids include, but are not limited to, plasmid DNA or nucleotide sequences encoding proteins, single or double stranded synthetic sequences, missense, antisense, nonsense and inhibitory RNAs that generate on and off, Mention may also be made of rate-regulating nucleotides that control the production of proteins, peptides and nucleic acids. Furthermore, nucleic acids can include, but are not limited to, genomic DNA, cDNA, siRNA, shRNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic or semi-synthetic sequences, and naturally and artificially derived sequences. In one embodiment, the nucleotide sequence can also include one that encodes the synthesis or inhibition of a therapeutic protein. Examples of such therapeutic proteins include, but are not limited to, anticancer agents, growth factors, hypoglycemic agents, antiangiogenic agents, bacterial antigens, viral antigens, tumor antigens or metabolic enzymes. Examples of anticancer agents include interleukin-2, interleukin-4, interleukin-7, interleukin-12, interleukin-15, interferon-α, interferon-β, interferon-γ, colony stimulating factor, Granulocyte macrophage stimulating factor, anti-angiogenic drug, tumor suppressor gene, thymidine kinase, eNOS, iNOS, p53, p16, TNF-α, Fas ligand, mutated oncogene, tumor antigen, viral antigen or bacterial antigen be able to. In another embodiment, the plasmid DNA can encode a shRNA molecule designed to inhibit protein (s) involved in the growth or maintenance of tumor cells or other hyperproliferative cells. Further, in some embodiments, plasmid DNA can simultaneously encode a therapeutic protein and one or more shRNAs. In other embodiments, the nucleic acid can be a mixture of plasmid DNA and synthetic RNA, including sense RNA, antisense RNA, ribozymes, and the like. Furthermore, nucleic acids can be variable in size, ranging from oligonucleotides to chromosomes. These nucleic acids can be derived from humans, animals, vegetables, bacteria, viruses or synthesis. These can be obtained by any technique known to those skilled in the art.

  The term “concentrated” as used herein refers to a composition whose dilution has been reduced. In some embodiments of the invention, the “concentrated” composition contains the condensed DNA, preferably in an isotonic solution. In certain embodiments of the invention, the concentrated composition comprises at least about 0.5 mg / ml of condensed DNA suspended in an isotonic solution.

  As used herein, the term “polymer backbone” is used to refer to a collection of polymer backbone molecules having a weight average molecular weight within a specified range. Thus, when describing that a molecule such as cholesterol is covalently bound thereto within a molar ratio range, such a ratio is the average number of cholesterol molecules bound to a collection of polymer backbone molecules. It should be understood to represent. For example, if cholesterol is described as being covalently attached to the polymer backbone at a molar ratio of 0.5, on average, cholesterol is attached to one-half of the polymer backbone molecule. As another example, if cholesterol is described as being covalently attached to the polymer backbone at a molar ratio of 1.0, on average, one cholesterol molecule is attached to each of the polymer backbones. . However, in reality, in this case, no cholesterol molecule is bonded to some polymer backbone molecules, while a plurality of cholesterol molecules may be bonded to other polymer backbone molecules. It should be understood that is the average number of bound cholesterol molecules from which the ratio is derived. The same logic applies to the molar ratio of polyethylene glycol to polymer backbone.

  As used herein, the term “peptide” is used to refer to a natural or synthetic molecule that contains two or more amino acids linked to the α-amino group of another amino acid by the carboxyl group of one amino acid. Can do. The peptides of the present invention are not limited by length, and therefore “peptides” can include polypeptides and proteins.

  As used herein, the terms “covalent” and “covalently” refer to chemical bonds in which electrons are shared between pairs of atoms.

  As used herein, a plurality of items, structural elements, compositional elements and / or materials may be presented in a common list for convenience. However, such an enumeration should be interpreted as each enumerated element being individually identified as a separate and unique element. Thus, unless stated otherwise, individual elements of such enumerations are effectively in effect on any other element listed in the same group, only on the basis that they are presented in a common group. Should not be construed as equivalent.

  Concentrations, amounts and other numerical data may be expressed or presented herein in a range format. It should be understood that such a range format is merely used for convenience and brevity, and therefore not only the numerical values explicitly listed as the upper and lower limits of the range, but also the range. All individual values or subranges contained within are to be construed flexibly as being included as if explicitly recited. For example, a numerical range of “about 1 to about 5” is interpreted to include not only the explicitly listed values of about 1 to about 5, but also individual values and subranges within the indicated range. Should. Therefore, this numerical range includes individual values such as 2, 3 and 4 and subranges such as 1 to 3, 2 to 4 and 3 to 5, and 1, 2, 3, 4 and 5 individually. It is. The same principle applies to ranges where only one number is listed as the minimum or maximum. Furthermore, such an interpretation shall apply regardless of the breadth or characteristics of the stated range.

  The present invention provides for highly enriching a low concentration nucleic acid composition (eg, 0.15 mg / ml) without affecting the physicochemical or biological properties of the nucleic acid or nucleic acid composition. Provide a method that can In one embodiment, the nucleic acid composition can be concentrated 33-fold or more without affecting these properties. Such highly concentrated nucleic acid compositions were extremely difficult in vivo, as previous attempts to achieve concentrations above about 0.3 mg / ml were extremely difficult due to low stability problems. Allows a wide range of dosing regimens.

  More specifically, the present invention provides concentrated and stable pharmaceutical compositions, including methods for the preparation and use of such compositions. For example, in one embodiment, a pharmaceutical composition comprising at least about 0.5 mg / ml of nucleic acid, wherein the nucleic acid is complexed with a cationic lipopolymer, and the complex is suspended in an isotonic solution. Provided. A complex suspended in an isotonic solution contains partially or fully condensed nucleic acid molecules. Cationic lipopolymers contain a cationic polymer backbone to which cholesterol and polyethylene glycol groups (ie, molecules) are covalently bonded. The molar ratio of cholesterol molecule to cationic polymer backbone is in the range of about 0.1 to about 10, and the molar ratio of polyethylene glycol molecule to cationic polymer backbone is in the range of about 0.1 to about 10. . In another embodiment, the molar ratio of polyethylene glycol molecules to cationic polymer backbone in the cationic lipopolymer is within the range of about 1 to about 10. In yet another embodiment, the molar ratio of polyethylene glycol molecules to cationic polymer backbone in the cationic lipopolymer is in the range of about 1 to about 5. In further embodiments, the molar ratio of cholesterol molecules to cationic polymer backbone in the cationic lipopolymer is in the range of about 0.3 to about 5. In yet another embodiment, the molar ratio of cholesterol molecules to cationic polymer backbone in the cationic lipopolymer is in the range of about 0.4 to about 1.5.

  The composition further comprises a filler excipient. The resulting composition is suitable for delivering nucleic acid to target cells and eliciting, inhibiting or modifying a biological response depending on the function of the nucleic acid.

  In one embodiment, the cholesterol and polyethylene glycol molecules can be directly covalently linked independently of the cationic polymer backbone. In another embodiment, the cholesterol and polyethylene glycol molecules are each indirectly covalently linked to the cationic polymer backbone. For example, a cholesterol molecule can be directly or indirectly attached to a polyethylene glycol molecule via a linker or spacer, and the polyethylene glycol molecule is covalently attached to the cationic polymer backbone. Alternatively, cholesterol molecules can be directly attached to the cationic lipopolymer backbone, and polyethylene glycol molecules can be indirectly attached to the lipopolymer via a linker or spacer.

  The specific linker between the polyethylene glycol and the cationic polymer backbone is an alkylene group having a terminal carboxy group, preferably a linear alkylene having 1 to 20 carbon atoms, more preferably about 2 to about 4 carbon atoms. It is a group. The terminal carboxy group on the linker forms an amide bond between the cationic lipopolymer and polyethylene glycol when attached to the amino group of the cationic polymer backbone. A suitable starting polyethylene glycol for reacting with the cationic polymer backbone molecule is a polyethylene glycol having a linker molecule terminated by an activating group, such as an N-hydroxysuccinimidyl ester. One example of such a polyethylene glycol is methoxypolyethylene glycol-propionic acid N-hydroxysuccinimidyl ester.

Some examples of cationic lipopolymer structures resulting from the reaction between polyethyleneimine, cholesteryl chloroformate (stereochemistry omitted) and methoxypolyethyleneglycol-propionic acid N-hydroxysuccinimidyl ester are the following structures: is there. The graphical representation reflects the approximate distribution of primary, secondary, and tertiary amino groups in polyethyleneimine, which is not present here for clarity, but that polyethyleneimine chains are regular. Assumes.

  In various aspects of the invention, n is typically from about 8 to about 20, more preferably from about 10 to about 15, and even more preferably about 12, and x is usually from about 2 to about 3, More specifically, it is about 2.5, y is typically from about 6 to about 10, more preferably from about 7 to about 9, and even more preferably 7.5, and z is usually about 0. .4 to about 0.8, more specifically about 0.5 to about 0.7, and even more limited to about 0.6.

  Furthermore, in some embodiments, a secondary condensation system is used to further condense nucleic acids that are already condensed using the techniques presented herein to stabilize nucleic acids at high concentrations. Can be further increased. Thus, prior to condensation according to embodiments of the invention, the nucleic acid can be in a partially condensed or non-condensed form. Secondary condensation systems include any condensation known to those skilled in the art including, but not limited to, cationic lipids, cationic peptides, cyclodextrins, cationized gelatin, dendrimers, chitosan and combinations thereof. Use materials or techniques may be included.

  For compositions according to embodiments of the invention, various degrees of condensation of nucleic acids can be achieved. In one embodiment, all nucleic acids or the majority of nucleic acids in the composition are condensed by forming a complex with a cationic polymer. In another embodiment, about 30% by weight of the nucleic acid in the composition is condensed. In yet another embodiment, about 50% by weight of the nucleic acid in the composition is condensed. In a further embodiment, about 70% by weight of the nucleic acid in the composition is condensed. In yet another embodiment, 90% by weight of the nucleic acid is condensed.

  Furthermore, the concentration of nucleic acid in the composition will vary depending on the materials used in the composition, the concentration method, and the intended use of the nucleic acid. However, in one embodiment, the concentration of nucleic acid is at least about 0.5 mg / ml. In another embodiment, the concentration of nucleic acid is at least about 1 mg / ml. In yet another embodiment, the concentration of nucleic acid is at least about 3 mg / ml. In further embodiments, the concentration of the nucleic acid can be at least about 5 mg / ml. In yet another aspect, the concentration of nucleic acid can be at least about 10 mg / ml. In another embodiment, the concentration of nucleic acid can be at least about 20 mg / ml. In yet another embodiment, the concentration of nucleic acid can be from about 10 mg / ml to about 40 mg / ml.

  Various methods can be used to determine the degree of condensation of the nucleic acid composition. For example, in one embodiment, the composition can be electrophoresed to determine the extent to which the nucleic acid in the composition forms a complex with a cationic polymer added to the composition. The electrostatic attraction of the negatively charged nucleic acid relative to the positively charged cationic lipopolymer prevents the nucleic acid from moving through the agarose gel. Thus, according to electrophoresis, the condensed nucleic acid by forming a complex with the cationic polymer remains immobile in the gel, whereas it is associated with the non-condensed nucleic acid, ie the cationic polymer. No nucleic acid travels a distance corresponding to the force of the current in the gel. In another example, nucleic acid condensation can be determined by particle size within the composition. The particle size can be measured by dynamic light scattering. Usually, a condensed nucleic acid has a smaller particle size than a non-condensed nucleic acid. Preferred condensed nucleic acids are those in which the nucleic acid and cationic lipopolymer nanoparticles have a size of about 50 nm to about 300 nm, more preferably about 50-200, and even more preferably about 50-150 nm.

  Any known nucleic acid can be utilized in the compositions and methods according to aspects of the invention, including the examples described above. Accordingly, the nucleic acids described herein should not be construed as limiting. For example, in one embodiment, the nucleic acid can include a plasmid encoding a protein, polypeptide or peptide. Many peptides are known which show benefit when formed as pharmaceutical compositions according to embodiments of the present invention. Without being limited thereto, some examples of such peptides include interleukin-2, interleukin-4, interleukin-7, interleukin-12, interleukin-15, interferon-α, Interferon-β, interferon-γ, colony stimulating factor, granulocyte macrophage colony stimulating factor, angiogenic agent, coagulation factor, hypoglycemic agent, apoptotic factor, anti-angiogenic agent, thymidine kinase, p53, IP10, p16, TNF-α , Fas ligand, tumor antigen, neuropeptide, viral antigen, bacterial antigen and combinations thereof. In one specific embodiment, the nucleic acid can be a plasmid encoding interleukin-12. In another embodiment, the nucleic acid can be a plasmid encoding an inhibitory ribonucleic acid. In yet another embodiment, the nucleic acid can be a short synthetic interfering ribonucleic acid. In a further embodiment, the nucleic acid is an antisense molecule designed to inhibit the expression of a therapeutic peptide.

  As mentioned above, the cationic lipopolymer can include a cationic polymer backbone to which cholesterol and polyethylene glycol are covalently bonded. The cationic polymer backbone can include any cationic polymer known to those of skill in the art that can be used to condense and concentrate nucleic acids according to various aspects of the invention. However, in one embodiment, the cationic polymer backbone includes polyethyleneimine, poly (trimethyleneimine), poly (tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine , Poly (2-dimethylamino) ethyl methacrylate, poly (lysine), poly (histidine), poly (arginine), cationized gelatin, dendrimer, chitosan and combinations thereof. In one specific embodiment, the cationic polymer backbone can be polyethyleneimine.

  In certain embodiments, the lipopolymer consists of polyethyleneimine (PEI) independently covalently linked to cholesterol and polyethylene glycol. In this embodiment, the average molar ratio of cationic lipopolymer PEG: PEI: cholesterol is about 2-3: 1: 0.25-1, preferably about 2.25-2.75: 1: 0.4. ~ 0.8, more preferably about 2.5: 1: 0.6. In certain embodiments, the molecular weight of such lipopolymer is about 3-4 kD (as the free base), preferably about 3.25-3.75 kD, more preferably about 3.54 kD, and the corresponding hydrochloride salt The molecular weight is about 4-5 kD, preferably about 4.5 kD.

  Furthermore, the molecular weight of the cationic polymer backbone can vary depending on many factors, including the nature of the nucleic acid, the intended use of the composition, and the like. However, in one embodiment, the molecular weight of the cationic polymer backbone can be from about 100 to about 500,000 daltons. In addition, the molecular weight of various other components of the cationic lipopolymer can also vary. For example, in one embodiment, the molecular weight of polyethylene glycol can be from about 50 to about 20,000 daltons.

  In constructing the pharmaceutical composition of the present invention, the molar ratio (N: P ratio) of the amine nitrogen in the functional cationic lipopolymer to the phosphate in the nucleic acid is sufficient to condense and / or concentrate the nucleic acid. It has been discovered that it can affect as much as possible. While the optimal N: P ratio may vary somewhat depending on the chemical properties of the nucleic acid, in one embodiment, the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is about 0.1: 1 to about 100: 1. In another embodiment, the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 3: 1 to about 20: 1. In yet another embodiment, the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 6: 1 to about 15: 1. In other embodiments, the ratio of amine nitrogen to phosphate in the nucleic acid is from about 3: 1 to about 100: 1, or from about 5: 1 to about 100: 1, or from about 7: 1 to about 100: 1. is there. In yet another embodiment, the ratio is from about 10: 1 to about 100: 1, or more preferably from 10: 1 to about 20: 1. In one specific embodiment, the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is about 11: 1.

  Further, it is also contemplated to include a filler excipient in the pharmaceutical composition. Such fillers can impart various beneficial properties to the formulation, such as cryoprotection, binding, isotonic balance, stabilization during lyophilization and reconstitution. It should be understood that the filler material can vary from composition to composition and the particular filler used should not be seen as being limited thereto. For example, in one embodiment, filler excipients can include various sugars, sugar alcohols, starches, celluloses, and combinations thereof. In another embodiment, the filler excipient is lactose, sucrose, trehalose, dextrose, galactose, mannitol, maltitol, maltose, sorbitol, xylitol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin, hydroxymethyl starch , Gelatin, sorbitol, ficol, sodium chloride, calcium phosphate, calcium carbonate, polyethylene glycol and combinations thereof. In yet another embodiment, the filler excipient is lactose, sucrose, trehalose, dextrose, galactose, mannitol, maltitol, maltose, sorbitol, xylitol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin and combinations thereof Can be included. In one specific embodiment, the filler excipient can comprise sucrose. In another specific embodiment, the filler excipient can comprise lactose.

  The concentration of the filler excipient in the composition is from about 0.01% to about 5%, more strictly from about 0.1% to about 3.0%, and even more strictly from about 0.1%. % To about 0.3%.

In some embodiments, it may be beneficial to functionalize the cationic lipopolymer to allow targeting of specific cells or tissues in the subject or culture. Such targeting is well known and the examples described herein should not be seen as limiting. For example, in one embodiment, the cationic lipopolymer can include a targeting moiety that is covalently bound to either the cationic lipopolymer or a polyethylene glycol molecule. Such targeting moieties can allow the cationic lipopolymer to circulate systemically throughout the subject's body to find and specifically target specific cell types or tissues. Examples of such targeting moieties include transferrin, asialoglycoprotein, antibody, antibody fragment, low density lipoprotein, cell receptor, growth factor receptor, cytokine receptor, folate, transferrin, insulin, asialo-olosomcoid, Mannose-6-phosphate, mannose, interleukin, GM-CSF, G-CSF, M-CSF, stem cell factor, erythropoietin, epidermal growth factor (EGF), insulin, asialo-orosomucoid, mannose-6-phosphate , mannose, Lewis ^X and sialyl Lewis ^X, N-acetyllactosamine, folate, galactose, lactose and thrombomodulin, fusogenic agents such as polymixin B and hemaglutinin HA2, rye Soso Motoro Fick factor (lys somotrophic agent), it can include a nuclear localization signal (NLS), and combinations thereof, such as T- antigen. The selection and binding of specific targeting moieties is well within the knowledge of those skilled in the art.

  The present invention also provides a lyophilized pharmaceutical composition that can be stored for an extended period of time and reconstituted prior to use. In one embodiment, the lyophilized pharmaceutical composition has a cationic lipopolymer comprising a cationic polymer backbone having cholesterol and polyethylene glycol covalently bound thereto, wherein the molar ratio of cholesterol to cationic polymer backbone is Filler excipients condensed with a cationic lipopolymer having a molar ratio of polyethylene glycol to cationic polymer backbone in the range of about 0.1 to about 10 and in the range of about 0.1 to about 10. And a lyophilized mixture of nucleic acids. Lyophilized pharmaceutical compositions can take a wide variety of forms, from dry powders to partially reconstituted mixtures.

  The present invention also includes methods for making various pharmaceutical compositions comprising condensed nucleic acids. For example, in one embodiment, a method of making a pharmaceutical composition having a condensed nucleic acid concentrated to at least 0.5 mg / ml in an isotonic solution is provided. In such a method, the cationic lipopolymer comprises a cationic polymer backbone to which cholesterol and polyethylene glycol are covalently bound, and the molar ratio of cholesterol to cationic polymer backbone is in the range of about 0.1 to about 10. And mixing the nucleic acid and the cationic lipopolymer in a filler excipient, wherein the molar ratio of polyethylene glycol to cationic polymer backbone is in the range of about 0.1 to about 10. The mixture can be lyophilized to a powder to concentrate the nucleic acid mixture, and later using a diluent to form a solution containing at least about 0.5 mg / ml condensed nucleic acid in an isotonic solution. Can be reconfigured to form.

  In general, the composition can be obtained by mixing a nucleic acid solution with a cationic lipopolymer solution in the presence of a disaccharide, followed by lyophilization and reconstitution in an isotonic solution. This process produces highly concentrated nucleic acid formulations that are scalable and have an extended shelf life, from several milligrams (bench scale) to several thousand milligrams (GMP scale). As described above, the cationic lipopolymer has a cationic polymer backbone in which polyethylene glycol and cholesterol are bound by a covalent bond. In the case of a polyethyleneimine skeleton, in one embodiment, the stoichiometry between polyethylene glycol and polyethyleneimine and between cholesterol and polyethyleneimine is in the range of 0.5 to 10, 0.1 to 10, respectively. . The chemical composition of the cationic polymer may be important to obtain a highly concentrated and stable nucleic acid formulation. Cationic polymers that do not show cholesterol and PEG linkage are less likely to produce stable, highly concentrated formulations, as shown in the examples below.

  Compositions according to embodiments of the present invention can also be combined with other condensed complexes of nucleic acids to achieve higher stability of the complex at high nucleic acid concentrations. For example, the addition of varying amounts of PEG-PEI-cholesterol can increase the stability of other nucleic acid delivery systems that are usually unstable at high nucleic acid concentrations.

  In various embodiments, the synthetic delivery system includes nucleic acids and cationic carriers that can be prepared by various techniques available in the art. Numerous cationic carriers for nucleic acids are known. For example, polyethyleneimine, poly (trimethyleneimine), poly (tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly (2-dimethylamino) ethyl methacrylate, poly (Lysine), poly (histidine), poly (arginine), cationized gelatin, dendrimer, chitosan, cationic lipids such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N- [1- ( 2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), 1- [2- (oleoyloxy) ethyl] -2-oleyl-3- (2-hydroxyethyl) Imidazolinium Lido (DOTIM), 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), 3β- [N- (N ′ , N′-dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-cholesterol HCl) diheptadecylamide glycylspermidine (DOGS), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N -(1,2-Dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N, N-dioleyl-N, N-dimethylammonium chloride DODAC) and their Combinations are mentioned. Combining these delivery systems with PEG-PEI-cholesterol increases the stability of the nucleic acid delivery system.

  Aspects of the invention also provide methods of using the pharmaceutical composition. For example, in one embodiment, a method of transfecting a mammalian cell comprises contacting the mammalian cell with a composition described herein, wherein the mammalian cell enters the cell and the biological activity of the nucleic acid is increased. Incubating under conditions that allow withdrawal can also be included. Such transfection techniques are known to those skilled in the art. Furthermore, in another embodiment, the target tissue can be transfected by delivering the composition to a warm-blooded organism or subject. Such delivery can be by dosage form such as intratumoral, intraperitoneal, intravenous, intraarterial, intratracheal, intrahepatic portal, oral, intracranial, intramuscular, intraarticular and combinations thereof. Such target tissues can include any tissue or subset of tissues that can benefit from transfection. Examples of such target tissues include, but are not limited to, ovary, uterus, stomach, colon, rectum, bone, blood, intestine, pancreas, chest, head, neck, lungs, spleen, liver, The kidney, brain, thyroid, prostate, bladder, thyroid, skin, abdominal cavity, thoracic cavity, and combinations thereof may be included.

  The following examples are provided to facilitate a clearer understanding of specific embodiments of the invention and are not intended to impose any limitations on the invention.

Preparation A
1 gram of branched polyethyleneimine (PEI) 1800 Da (0.56 mM) is dissolved in 5 ml of chloroform, placed in a ml round bottom flask and stirred at room temperature for 20 minutes. 380 mg of cholesteryl chloroformate (0.84 mM) and 500 mg of activated methoxypolyethylene glycol (MPEG-SPA, methoxypolyethyleneglycol-propionic acid N-hydroxysuccinimidyl ester) (550 Da) (0.91 mM) in 5 ml of chloroform Dissolve and transfer to addition funnel placed on top of round bottom flask containing PEI solution. A mixture of cholesteryl chloroformate and MPEG-SPA in chloroform is slowly added to the PEI solution over 5-10 minutes at room temperature. The solution is further stirred at room temperature for 4 hours. After removing the solvent with a rotary evaporator, the remaining sticky substance is dissolved in 20 ml of ethyl acetate with stirring. The product is precipitated from the solvent by slowly adding 20 ml of n-hexane and the liquid is poured gently from the product. The product is washed twice with a 20 ml mixture of ethyl acetate / n-hexane (1/1; v / v). After pouring the liquid gently, the material is dried by purging with nitrogen gas for 10-15 minutes. Dissolve the material in 10 ml 0.05 N HCl to prepare the salt form of the amine group. The aqueous solution is filtered through 0.2 μm filter paper. The final product is obtained by lyophilization.

  The molar ratio of the preparation in this example conjugates 3.0 moles of MPEG-SPA and 1.28 moles of cholesterol to 1 mole of PEI molecule.

Preparation B
Mix 20 grams (11.1 mmol) of branched PEI (BPEI) and 200 mL of dry chloroform together to dissolve the BPEI. After dissolution, 18.7 g (26 mmol) of cholesteryl chloroformate 4 g and activated methoxypolyethylene glycol (MPEG-SPA methoxypolyethylene glycol-propionic acid N-hydroxysuccinimidyl ester, MPEG MW550, ester MW719) in 200 mL of dry chloroform. The containing solution is added dropwise to the reaction mixture with stirring over 20-30 minutes, followed by an incubation period of 3-4 hours. The mixture is then left under vacuum and the solution is concentrated to remove residual chloroform. The resulting residue is dissolved in 320 mL of 1M aqueous HCl and stirred. This solution of PPC hydrochloride is again concentrated under vacuum to give a very sticky material. To isolate PPC hydrochloride and remove reaction by-products and unreacted starting material, the concentrated mixture is mixed with acetone (<0.4% water) and stirred to give PPC hydrochloride as a free-flowing material. Cause precipitation. After precipitation, discard the supernatant. Hygroscopic PPC hydrochloride is dried under vacuum.

  A polymer DNA complex is first generated by preparing PPC and DNA at an appropriate concentration in 10% lactose. A stock solution of cationic polymer (5 mg / ml) and DNA (3 mg / ml) in water for injection is diluted with 0.3-3% lactose solution. These are required upon reconstitution to achieve the final 10% lactose concentration. Next, DNA is added dropwise to the PPC solution with stirring and incubated for 15 minutes at room temperature to form a complex.

  Add 500 μl of the prepared composition to a 2 ml borosilicate glass bottle and place in a freedryer. Cool the bottle to -34 ° C for 4 hours before starting primary drying. After 24 hours, the shelf temperature is raised to 20 ° C. and maintained under vacuum for an additional 24 hours. Finally, the shelf temperature is raised to 4 ° C. and the bottle is capped under vacuum.

Preparation C
180 milligrams of branched PEI 1800 (0.1 mM) is dissolved in 4 ml of chloroformate and stirred at room temperature for 30 minutes. 70 mg cholesteryl chloroformate (0.14 mM) and 48 mg PEG330 (0.14 mM) are dissolved in 1 ml chloroformate and slowly added to the PEI solution using a syringe over 3-10 minutes. The mixture is stirred at room temperature for 4 hours. For precipitation, after adding 10 ml of ethyl acetate, the solution is incubated overnight at −20 ° C. and then the liquid is gently poured from the flask. The residual material is washed twice with a 5 ml mixture of ethyl acetate / n-hexane (l / l; v / v). The residual material is dried by nitrogen purge for 10-15 minutes, dissolved in 10 ml 0.05N HCl for 20 minutes, and then the solution is filtered through a 0.2 μm syringe filter. The aqueous solution is freeze-dried by freeze drying to remove moisture from the polymer.

  The molar ratio of this preparation conjugates 0.85 mole of PEG and 0.9 mole of cholesterol to 1 mole of PEI molecule.

Preparation D
Dissolve 500 milligrams of 25 kDa linear PEI (0.02 mM) in 30 ml and stir for 30 minutes at 65 ° C. A three-necked flask is equipped with a condensation and addition funnel. A mixture of 200 mg mPEG-NHS1000 (0.2 mM) and 40 mg cholesteryl chloroformate (0.08 mM) in 5 ml chloroform is slowly added to the PEI solution over 3-10 minutes. The solution is continuously stirred for an additional 4 hours at 65 ° C. and the volume is reduced to about 5 ml on a rotary evaporator. The solution is precipitated into 50 ml of ethyl ether, free cholesterol is removed, the liquid is poured gently from the flask and the remaining material is washed twice with 20 ml of ethyl ether. After drying with pure nitrogen, the material is dissolved in a mixture of 10 ml of 2.0 N HCl and 2 ml of trifluoroacetic acid. The solution is dialyzed against deionized water for 48 hours using a MWCO 15000 dialysis tube and replaced with fresh water every 12 hours. The solution is lyophilized to remove moisture.

  The molar ratio of this preparation conjugates 12.0 moles of PEG and 5.0 moles of cholesterol to 1 mole of PEI molecule.

Preparation E
1 gram of PEI (MW: 1200 Daltons) is dissolved in a mixture of 15 mL anhydrous methylene chloride and 100 μl triethylamine (TEA). After stirring on ice for 30 minutes, 1.2 g cholesteryl chloroformate solution is slowly added to the PEI solution and the mixture is stirred overnight on ice. The product obtained is precipitated by adding ethyl ether, then centrifuged and subsequently washed with additional ethyl ether and acetone. The water-insoluble lipopolymer is dissolved in chloroform to obtain a final concentration of 0.08 g / mL. Following synthesis and purification, MALDI-TOFF MS and ¹ H NMR are used to characterize the water-insoluble lipopolymer.

  NMR measurement of water insoluble lipopolymer 1200 shows that the amount of cholesterol conjugated to PEI is about 40%. MALDI-TOF mass spectroscopic analysis of a water-insoluble lipopolymer shows a molecular weight of about 1600.

Preparation F
3 grams of PEI (MW: 1800 Dalton) is stirred for 30 minutes on ice in a mixture of 10 ml of anhydrous ethylene chloride and 100 μl of triethylamine. 1 gram of cholesteryl chloroformate is dissolved in 5 ml of ice-cold anhydrous methylene chloride and then slowly added to the PEI solution over 30 minutes. The mixture is stirred for 12 hours on ice and the resulting product is dried on a rotary evaporator. Dissolve the powder in 50 ml of 0.1N HCl. The aqueous solution is extracted three times using 100 mL of methylene chloride, and then filtered through a glass microfiber filter. The product is concentrated by solvent evaporation, precipitated with a large excess of acetone and dried under vacuum. The product is analyzed using MALDI-TOF mass spectrophotometry and 10 ¹ H NMR. NMR results for water-soluble lipopolymer 1800 show that the amount of cholesterol conjugated to PEI is about 47%. PEACE's MALDI-TOFF mass spectrometric analysis shows a molecular weight of about 2200. This suggests that the molar ratio of cholesterol to PEI is 1/1 in the majority of PEACE1800, although some are not conjugated or conjugated at a molar ratio of 2/1 (cholesterol / PEI). To do.

Preparation G
50 milligrams of PEI 1800 is dissolved in 2 mL of anhydrous methylene chloride on ice. Next, 200 μL of benzyl chloroformate is slowly added to the reaction mixture and the solution is stirred on ice for 4 hours. After stirring, 10 mL of methylene chloride is added and the solution is extracted with 15 mL of saturated NH ₄ Cl. Remove the water from the methylene chloride phase using magnesium sulfate. The volume of the solution is reduced under vacuum and the product, CBZ protected PEI is precipitated using ethyl ether. 50 mg of primary amine CBZ protected PEI is dissolved in methylene chloride, 10 mg of cholesterol chloroformate is added and the solution is stirred on ice for 12 hours. The product, CBZ protected lipopolymer, is precipitated using ethyl ether, washed with acetone, and then in DMF containing palladium activated carbon as a catalyst under H ₂ as a hydrogen donor. Dissolve with. The mixture is stirred at room temperature for 15 hours, filtered through CELITE®, and the volume of the solution is reduced on a rotary evaporator. The final product is obtained from precipitation with ethyl ether.

Preparation H
NH ₂ -PEG-COOH3400 a (0.15 mM) 500 mg, 30 minutes at room temperature, was dissolved in anhydrous chloroform 5 ml. A solution of 676 mg of cholesterol chloroformate (1.5 mM) in 1 ml of anhydrous chloroform is slowly added to the PEG solution and then stirred for an additional 4 hours at room temperature. The mixture is precipitated for 1 hour on ice in 500 ml of ethyl ether and then washed three times with ethyl ether to remove unconjugated cholesterol. After drying with a nitrogen purge, the powder is dissolved in 5 ml of 0.05N HCl to acidify the carboxyl groups on the PEG. Dry the material with a freeze dryer. 100 milligrams of PEI 1800 (0.056 mM), 50 mg DCC and 50 mg NHS are dissolved in 5 ml chloroform at room temperature and the mixture is stirred for 20 minutes, after which a solution of 380 mg chol-PEG-COOH in 1 ml chloroform is slowly added to the PEI solution. After stirring at room temperature for 6 hours, the organic solvent was removed using a rotary evaporator. The residual material was dissolved in 10 ml of deionized water and purified by FPLC.

(Example 1)
Preparation of Condensed Nucleic Acid Concentrated Liquid Formulation with Cationic Lipopolymer This example describes the preparation of a highly concentrated formulation of fully condensed nucleic acid in bench scale production. This involves preparing a nucleic acid complex having a cationic polymer, followed by lyophilization and reconstitution into an isotonic solution. The nucleic acid used was plasmid DNA encoding IL-12 or a luciferase gene, and the polymer contained a polyethyleneimine (PEI) backbone covalently linked to polyethylene glycol (PEG) and cholesterol (Chol) (PEG- PEI-Chol or PPC). The molar ratios between PEG and PEI and between cholesterol and PEI are 0.5-10 and 0.1-10, respectively. First, DNA and PPC solutions are prepared separately at 5 mg / ml in water for injection and then diluted to 0.15 mg / ml (DNA) and 0.554 mg / ml (PPC) with 3% lactose. Using a micropipette, DNA in lactose solution was added to PPC in lactose solution at a nitrogen to phosphate ratio (N: P ratio) of 11: 1 and the formulation was incubated for 15 minutes at room temperature to form a complex. It can be so. PPC / DNA complexes in 3% lactose are lyophilized using a FREEZONE freeze-drying system from LABCONCO, Kansas City, Missouri. 500 μl of the prepared formulation was added to a 2 ml borosilicate glass bottle and then lyophilized using a freeze drying program consisting of the following segments:
1) Freezing segment (lamp at 0.25 ° C / min, hold at 34 ° C for 4 hours)
2) Primary drying segment (held at 34 ° C. for 24 hours)
3) Secondary drying segment (ramp to 20 ° C., hold for 24 hours) and 4) Ramp to 4 ° C. at 0.25 ° C./min.

  The resulting lyophilized powder is reconstituted with water for injection to various concentrations from 0.1 mg / ml to 20 mg / ml DNA. A representative batch of small scale preparations resulted in 100-200 mg of fully formulated DNA.

(Example 1A)
A nucleic acid / cationic lipopolymer formulation is prepared using a cationic lipopolymer and an IL-12 nucleic acid at an N: P ratio of 11: 1, essentially following the procedure outlined in Example 1 above. The cationic lipopolymer has a PEG: PEI: cholesterol molar ratio of about 2.5: 1: 0.6 and a molecular weight (free base) of about 3.54 kD. The resulting formulation containing lactose can be lyophilized and reconstituted to a nucleic acid concentration of at least about 0.5 mg / ml without nucleic acid aggregation or significant loss of transfection activity.

(Example 2)
Preparation of Condensed Nucleic Acid Concentrated Liquid Formulation With Cationic Lipopolymer This example describes the preparation of a highly concentrated formulation of condensed nucleic acid, as shown in FIG. This protocol produces over 6000 mg of fully formulated DNA (as opposed to 100-200 mg of DNA produced from the small scale preparation described in Example 1) and scales to higher yields Is possible. The scaled-up method uses a peristaltic pump to mix large amounts of DNA and polymer solutions, achieve an online mixing scenario, form a complex, and then perform a freeze-dry cycle that can accommodate large volumes. Accompanied. Briefly, DNA and PPC solutions are prepared in 3% lactose at 0.3 mg / ml and 1.1 mg / ml, respectively. Using a peristaltic pump (WATSON MARLOW, SCI400) with an internal diameter of a silicon tube (WATSON MARLOW, Z982-0088) of 0.89 mm, the two components are combined at a constant flow rate of 225 ± 25 ml / min. The two mixtures are joined using a polypropylene T connector at the end of each tube. Nanoparticles formed immediately upon mixing of the polymer and DNA solution. 40 ml of the formulated complex is placed in a 100 ml glass bottle and lyophilized using a freeze drying program consisting of the following segments:
1) Pre-freeze at -50 ° C for up to 720 minutes,
2) Primary drying at 65 μm Hg for a maximum of 180 minutes at −40 ° C., then a maximum of 1980 minutes at −34 ° C.
3) Secondary drying at 65 μm Hg for up to 720 minutes at −25 ° C., for up to 3180 minutes at −15 ° C., for up to 1500 minutes at −10 ° C., and at maximum 1440 minutes at 4 ° C.

  Reconstitute the lyophilized powder obtained in various concentrations from 0.1 mg / ml to 20 mg / ml DNA using water for injection. A typical batch of this scale will have a fully formulated DNA amount of 6000 mg.

(Example 3)
Determination of the particle size of a concentrated liquid formulation of condensed nucleic acid with cationic lipopolymer A highly concentrated formulation of plasmid DNA with cationic lipopolymer, ie PPC, is described in Examples 1 and 2. Prepare as follows. For determination of polymer / nucleic acid particle size, aliquots of liquid formulations are analyzed using a 90 Plus / BI-MAS Particle Sizer from BROOKHAVEN INSTRUMENTS, Holtsville, NY. Specifically, for analysis, 50 μl of the formulation is added to 950 μl of milli-Q water in a polystyrene cuvette.

  FIG. 2 shows that the DNA / PPC complex in pre-dried frozen or non-concentrated formulation (0.15 mg / ml DNA) and IL-12 at higher concentrations from 0.5 mg / ml to 10 mg / ml. The particle size of the DNA / PPC complex after reconstitution using plasmid (FIG. 2A) or luciferase plasmid (FIG. 2B) is shown. Reconstitution at higher concentrations has no significant effect on particle size, suggesting that the complex is stable.

(Example 4)
Analysis of Nucleic Acid Condensates of Concentrated Liquid Formulations of Nucleic Acids Having Cationic Lipopolymer In this example, the ability of PPC polymers to condense plasmid DNA is evaluated. A highly concentrated formulation of plasmid DNA with cationic lipopolymer, ie PPC, is prepared as described in Examples 1 and 2. The nucleic acid / polymer complex is electrophoresed using a 1% agarose gel. Due to the electrostatic attraction of the negatively charged plasmid DNA relative to the positively charged PPC polymer, the DNA cannot move through the agarose gel. As shown in FIG. 3, any DNA present in the highly concentrated formulation is condensed.

(Example 5)
Determination of Nucleic Acid Concentration of Concentrated Liquid Formulations of Nucleic Acids Containing Cationic Lipopolymers Using an AGILENT 8453 spectrophotometer (AGILENT TECHNOLOGIES, Santa Clara, Calif.), In highly concentrated formulations in DNA and PPC complexes Measure the amount of nucleic acid. Dilute 50 μl of formulation using 950 μl of water for injection (WFI) in a quartz cuvette and measure the absorbance rate using a wavelength of 260 nm. Assuming 1 optical density (at 260 nm) = DNA 50 μg / ml, the DNA concentration is measured.

(Example 6)
Determination of Transfection Activity of Concentrated Liquid Formulations of Nucleic Acids Having Cationic Lipopolymers The transfection activity of highly concentrated formulations of DNA and PPC complexes is measured in vitro. Perform a direct comparison with the unconcentrated formulation. Transfection complexes containing luciferase or IL-12 plasmid are prepared by the method described in Examples 1 and 2 and reconstituted at DNA concentrations from 0.15 mg / ml to 10 mg / ml. Cos-1 cells (1.5 × 10 ⁵ cells / well) are seeded in 12-well tissue culture plates in 10% fetal bovine serum (FBS). Incubate each well for 6 hours with 4 μg of complexed DNA in a minimal essential medium (DMEM) from Dulbecco / Vog Modified Eagle in a total volume of 500 μl in the absence of FBS. At the end of the incubation period, supplement with 10% FBS for an additional 40 hours and replace the medium with 1 ml of fresh DMEM. At the end of the incubation period, transfection activity was measured in cell culture medium (IL-12) or cell lysate (luciferase). For measurement of IL-12 levels, the cell culture medium is analyzed directly by IL-12 ELISA analysis. For luciferase measurement, cells are washed with phosphate buffered saline and lysed with TENT buffer (50 mM Tris-Cl [pH 8.0] 2 Mm EDTA, 150 mM NaCl, 1% Triton X-100). To do. The Orion Microplate Luminometer (BERTHOLD DETECTION SYSTEMS, Oak Ridge, TN) is used to measure luciferase activity in cell lysates as relative light intensity values (RLU). The final value of luciferase is reported in units of RLU / total protein (mg). Total protein levels are measured using a BCA protein analysis kit (PIERCE BIOTECHNOLOGY, Rockford, Ill.). The levels of IL-12 and luciferase expression from highly concentrated formulations of IL-12 and luciferase plasmid / PPC complex are shown in FIGS. 4A and 4B, respectively. The data shows that the transfection activity of the nucleic acid complex in a highly concentrated form is preserved.

(Example 7)
Evaluation of various excipient sugars and their properties in the preparation of concentrated liquid formulations of nucleic acids with cationic lipopolymers Preparation of highly concentrated formulations of two commonly used sugars, lactose and sucrose Are evaluated as candidates for expansion agents or fillers that can be used during the freeze-drying process. PPC / DNA complexes are prepared at 3%, 1.5% and 0.3% with lactose and sucrose, respectively. Using the protocol of Example 1, the formulation is lyophilized. Following the freeze-drying step, the formulation is reconstituted using WFI and reconstituted with final DNA concentrations of 0.5 mg / ml, 1 mg / ml and 5 mg / ml. Particle size and in vitro gene transfer are evaluated for these various formulations. As shown in Table 1, both the particle size and transfection activity are preserved regardless of whether the antifreeze filler is sucrose or lactose. These results indicate that two or more sugars can be used for the purpose of preparing highly concentrated physicochemically and biologically stable nucleic acids having cationic polymers.
Table 1
Evaluation of excipient sugars in the preparation of concentrated isotonic formulations of nucleic acids with cationic lipopolymers

(Example 8)
IL-12 expression in normal brain parenchyma after intracranial expression of concentrated liquid formulations of nucleic acids with cationic lipopolymers Is highly concentrated formulations of nucleic acids and cationic lipopolymers biologically active in vivo? To determine whether the IL-12 plasmid with a cationic polymer, ie, PPC, is administered directly to normal brain tissue. After treatment, IL-12 immunohistochemical staining is performed on brain slices from animals euthanized 14 days or 1 month later. The brain parenchyma of animals treated with PPC alone did not show any IL-12 staining (FIG. 5A). In contrast, the brain parenchyma of mice injected with pmIL-12 / PPC showed positive staining for IL-12 in the cranium (FIG. 5B). This experiment demonstrates that the biological activity of nucleic acid complexes with cationic polymers is preserved during the concentration process. In addition, it can be concluded that cytokines remain present for at least one month after infusion. Furthermore, the presence of this cytokine in the brain of animals that survived to euthanization suggests that the actual expression of IL-12 does not cause lethal toxicity in the brain.

(Example 9)
Efficacy of concentrated liquid formulation of nucleic acid with cationic lipopolymer in mouse glioma model Anti-cancer effect of highly concentrated formulation of fully complexed nucleic acid expressing IL-12 gene Are examined in a mouse glioma model. An intracranial injection of 1 × 10 ⁵ GL261 glioma cells was performed along with a co-injection of 3 μl of IL-12 / PPC complex from a highly concentrated formulation of 5 mg / ml IL-12 plasmid DNA. Implant the tumor into the mouse cerebral cortex. Animals are monitored for signs of neurotoxicity and, when possible, the cadaver is dissected to confirm that the cause of death is an intracranial tumor. Plot survival using Kaplan-Meier survival analysis. A single intracranial injection of the pmIL-12 / PPC complex administered at a plasmid dose of 15 μg is well tolerated since no significant adverse events have been observed. A single injection of pmIL-12 / PPC complex at a plasmid dose of 15 μg showed results that significantly improved animal survival (FIG. 6).

(Example 10)
Biological activity of concentrated liquid formulation of nucleic acid with cationic lipopolymer in patients with ovarian cancer The biological activity of highly concentrated formulation of fully condensed nucleic acid expressing IL-12 gene can be Investigate in cancer patients. As a result of four weekly intraperitoneal administrations of highly concentrated isotonic formulations of IL-12 plasmid and PPC to women suffering from recurrent ovarian cancer, , A significant level of IL-12 surrogate marker, IFN-γ, occurred. IFN-γ levels vary from 20 to 275 pg per ml of ascites. These data demonstrate that highly concentrated formulations of IL-12 nucleic acid are suitable for clinical applications.

(Example 11)
Evaluation of the effect of the chemical composition of the cationic polymer on the properties of the concentrated liquid formulation of nucleic acids with cationic lipopolymer Concentration of nucleic acid formulations with cationic gene carriers such as lipids or polymers is less stable as a result of the concentration step Previous attempts have shown that this is extremely difficult because transfection is lost. The success in producing high concentrations of fully condensed nucleic acids that are physicochemical and biologically stable is whether it is inherent in the chemical composition of the test cationic polymer, PEG-PEI-cholesterol (PPC) To determine, other cationic polymers are tested, including free PEI, PEI conjugated to cholesterol or PEI conjugated to PEG and cationic liposomal DOTAP. DNA complexes are prepared at 0.15 mg / ml as described in Example 1 and then concentrated to 0.5 and 5 mg / ml. Particle size and transfection activity are measured as described in Examples 3 and 6. As shown in FIGS. 7 and 8, DNA complexes prepared using free PEI (PEI1800, PEI15000, PEI25000) or PEI-cholesterol, PEI-PEG or the cationic lipid DOTAP were lyophilized and 0.5 mg / ml Alternatively, after reconstitution to 5 mg / ml, aggregation and loss of transfection activity did not produce a stable complex. The destabilizing effect is more pronounced at 5 mg / ml than at 0.5 mg / ml. In contrast, DNA complexes prepared using PEG-PEI-cholesterol (PPC) maintain physicochemical and transfection properties during lyophilization and reconstitution at high DNA concentrations (FIG. 7). And 8). These results suggest that covalent modification of cationic polymers with cholesterol and PEG is essential for conservation of activity during the concentration process.

(Example 12)
Long-term stability of lyophilized or concentrated liquid formulations of nucleic acids with cationic polymers Large-scale lots of lyophilized IL-12 / PPC complexes were obtained using the method outlined in Example 2 And stored at −80 ° C., −20 ° C., 4 ° C. and 25 ° C. (60% RH) for stability evaluation. At the time of analysis, the bottle is removed from storage and 2.4 mL of WFI is added. For each sample, pH, DNA concentration, osmolarity, particle size and biological activity were measured. As shown in FIG. 9, the DNA concentration, pH, osmolality and particle size of the IL-12 / PPC complex are stored for 2 years at the indicated temperature. The gene transfer activity of pIL-12 / PPC is quantified in COS-1 cells as described in Example 6. COS-1 cells are transfected with 4 μg of DNA using biological material. The level of IL-12 in the cell culture medium is quantified 48 hours after transfection using a commercially available ELISA kit. The bioactivity results obtained from the 2-year stability study are shown in FIG. There is no significant change in the biological activity of the biological product during storage at -80 ° C or -20 ° C. At 0 hours, the activity is 151 ± 130 pg / mL, and with the passage of time, the remaining data varies within this standard deviation, except for 25 ° C., which always shows a decrease. At 4 ° C, a decrease in transfection activity is observed after 360 days, but there is no follow-up time to reach a conclusive evaluation due to lack of sample.

(Example 13)
Stability of reconstituted substances in concentrated liquid formulations of nucleic acids with cationic polymers The stability of the reconstituted substances is investigated in another study. Lyophilized IL-12 plasmid DNA / PPC complex is prepared according to the method described in Example 2 and reconstituted in water for injection to 0.5 mg / ml. Store the reconstituted material at 4 ° C. Samples are removed after 60 and 90 days and analyzed for particle size, osmolality and gene expression. Freeze-dried products placed in sealed bottles and stored at -80 ° C are analyzed simultaneously for comparison. As shown in Table 2, reconstituted EGEN-001 is stable at 4 ° C. for at least 90 days after reconstitution with WFI. Stability parameters, including DNA concentration, particle size, osmolarity or gene expression, do not change significantly compared to lyophilized material stored in sealed bottles and stored at -80 ° C.
Table 2
Long-term stability of reconstituted forms of highly concentrated, fully condensed, isotonic formulations of nucleic acids with cationic polymers at 4 ° C

(Example 14)
Preparation of highly concentrated and stable DNA formulation of synthetic nucleic acid delivery system by co-formulation with PEG-PEI-cholesterol PEG-PEI-cholesterol is added to an existing synthetic nucleic acid delivery system and at high nucleic acid concentrations , Usually increase the stability of unstable nucleic acid formulations.

In one example, PEG-PEI-cholesterol is added to a DNA formulation prepared using linear polyethyleneimine 25 kDa (LPEI 25 kDa). In the presence of 3% lactose, a DNA formulation at a concentration of 0.15 mg / ml can be prepared using 10: 1 (N: P ratio) LPEI 25 kD. PEG-PEI-cholesterol lipopolymer can then be added to the LPE125 kD / DNA complex at various PPC ratios relative to the formulated DNA. For example, PPC / DNA (N: P ratio) is (0: 1), (1: 1), (5: 1), (7.5: 1), (11: 1), (15: 1) And (20: 1). After adding 500 μl of each formulation to a 2 ml borosilicate glass bottle, it can be lyophilized in a freeze-dry system. The freeze-dry program consists of the following segments:
1) Freezing segment (lamp at 0.25 ° C / min, hold at -34 ° C for 4 hours),
2) Primary drying segment (held at -34 ° C for 24 hours),
3) Secondary drying segment (ramp to -20 ° C, hold for 24 hours) and 4) Ramp to 4 ° C at 0.25 ° C / min.

  The dry frozen formulation can be reconstituted to 0.5 mg / ml or other suitable concentration using water for injection.

  It should be understood that the compositions and application schemes described above are only intended to illustrate preferred embodiments of the present invention. Many modifications and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the invention, and the appended claims are intended to cover such modifications and arrangements. Is intended. Thus, while the present invention has been described in particular detail herein, with reference to what is presently considered to be the most practical and preferred embodiment of the invention, it is set forth herein. It is understood that many changes may be made, including, but not limited to, size, material, shape, type, function and method of operation, assembly and use variations without departing from the principles and concepts described. It is obvious to the contractor.

Claims (52)

(A) a mixture of a cationic lipopolymer suspended in an aqueous solution and at least about 0.5 mg / ml nucleic acid, wherein the cationic lipopolymer is independently covalently bonded to cholesterol and polyethylene glycol groups A mixture comprising a backbone, wherein the molar ratio of cholesterol to cationic polymer backbone is from about 0.1 to about 10, and the molar ratio of polyethylene glycol to cationic polymer backbone is from about 0.1 to about 10, and (b) A composition comprising a filler excipient.   The composition of claim 1, wherein the mixture of nucleic acid and lipopolymer forms a complex.   The composition of claim 1, wherein the aqueous solution is an isotonic solution.   The composition of claim 1 comprising a condensed nucleic acid.   2. The composition of claim 1, wherein at least about 30% by weight of the nucleic acid is condensed.   2. The composition of claim 1, wherein at least about 90% by weight of the nucleic acid is condensed.   2. The composition of claim 1 that can be dried and reconstituted to a nucleic acid concentration of at least 0.5 mg / ml without nucleic acid aggregation.   Cationic polymer backbone is polyethyleneimine, poly (trimethyleneimine), poly (tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly (2-dimethylamino) The composition of claim 1, wherein the composition is an element selected from the group consisting of ethyl methacrylate, poly (lysine), poly (histidine), poly (arginine), cationized gelatin, dendrimer, chitosan, and combinations thereof.   The composition of claim 1, wherein the concentration of the nucleic acid is at least 1 mg / ml.   The composition of claim 1, wherein the concentration of the nucleic acid is at least 10 mg / ml.   The composition of claim 1, wherein the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 0.1: 1 to about 100: 1.   Nucleic acid is interleukin-2, interleukin-4, interleukin-7, interleukin-12, interleukin-15, interferon-α, interferon-β, interferon-γ, colony stimulating factor, granulocyte macrophage colony stimulating factor , Angiogenic agent, coagulation factor, hypoglycemic agent, apoptotic factor, anti-angiogenic agent, thymidine kinase, p53, IP10, p16, TNF-α, Fas ligand, tumor antigen, neuropeptide, viral antigen, bacterial antigen and their 2. The composition of claim 1, which is a plasmid encoding a peptide selected from the group consisting of combinations.   The composition of claim 1, wherein the molecular weight of the cationic polymer backbone is from about 50 to about 500,000 daltons.   The composition of claim 1, wherein the molar ratio of polyethylene glycol to cationic polymer backbone in the cationic lipopolymer is in the range of about 1 to about 10.   The composition of claim 1, wherein the filler excipient is an element selected from the group consisting of sugar, sugar alcohol, starch, cellulose, and combinations thereof.   Filler excipients are lactose, sucrose, trehalose, dextrose, galactose, mannitol, maltitol, maltose, sorbitol, xylitol, mannose, glucose, fructose, polyvinylpyrrolidone, glycine, maltodextrin, hydroxymethyl starch, gelatin, sorbitol, ficoll 2. The composition of claim 1, wherein the composition is an element selected from the group consisting of sodium chloride, calcium phosphate, calcium carbonate, polyethylene glycol, and combinations thereof.   The composition of claim 1 wherein the filler excipient is sucrose.   The composition of claim 1, wherein the filler excipient is lactose.   The composition according to claim 1, wherein the nucleic acid is a plasmid encoding the interleukin-12 gene, and the cationic polymer backbone is polyethyleneimine (PEI).   20. The composition of claim 19, wherein the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 10: 1 to about 100: 1.   20. The composition of claim 19, wherein the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 11: 1 to about 20: 1.   The composition of claim 1, wherein the nucleic acid is a plasmid encoding an inhibitory ribonucleic acid.   The composition of claim 1, wherein the nucleic acid is a short synthetic interfering ribonucleic acid. A method of making a pharmaceutical composition comprising at least about 0.5 mg / ml of nucleic acid suspended in an isotonic solution comprising:
Mixing a nucleic acid, a cationic lipopolymer and a filler excipient in an aqueous solvent, the cationic lipopolymer comprising a cationic polymer independently covalently bonded to cholesterol and polyethylene glycol groups, The molar ratio is from about 0.1 to about 10 and the molar ratio of polyethylene glycol to the cationic polymer backbone is from about 0.1 to about 10.
Lyophilizing the mixture into a powder and reconstituting the powder with a diluent to form a solution containing at least about 0.5 mg / ml of condensed nucleic acid in an isotonic solution.   23. The method of claim 22, wherein the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 10: 1 to about 100: 1. A mixture of filler excipient, nucleic acid and cationic lipopolymer, wherein the cationic lipopolymer has at least one cholesterol molecule and at least one polyethylene glycol molecule independently covalently bound thereto And a mixture wherein the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 10: 1 to about 100: 1. Contacting the mammalian cell with the composition of claim 1 and incubating the mammalian cell under conditions that allow the composition of claim 1 to enter the cell and elicit the biological activity of the nucleic acid. Method for transfection of mammalian cells.   A method of transfection of a target tissue comprising delivering the composition of claim 1 to a warm-blooded organism.   The dosage form is further selected from the group consisting of intratumoral, intraperitoneal, intravenous, intraarterial, intratracheal, endohepatic, oral, intracranial, intramuscular, intraarticular and combinations thereof. 30. The method of claim 28, which can be included.   Target tissue is ovary, uterus, stomach, colon, rectum, bone, blood, intestine, pancreas, chest, head, neck, lung, spleen, liver, kidney, brain, thyroid, prostate, bladder, thyroid, skin, abdominal cavity 30. The method of claim 28, wherein said method is localized to an element selected from the group consisting of a thoracic cavity and combinations thereof. (A) a complex formed of a nucleic acid and a cationic lipopolymer, wherein the cationic lipopolymer has a polyethylene glycol: polyethyleneimine: cholesterol molar ratio in the range of about 2-3: 1: 0.25-1. A composite comprising a cationic polymer backbone covalently bonded independently to cholesterol and polyethylene glycol groups and suspended in an isotonic solution, and (b) a filler excipient.   32. The composition of claim 31, wherein the nucleic acid is interleukin-12.   The lipopolymer consists of polyethyleneimine (PEI) independently covalently bonded to cholesterol and polyethylene glycol, with an average molar ratio of PEG: PEI: cholesterol in the range of about 2-3: 1: 0.25-1. 33. The composition of claim 32.   34. The composition of claim 33, wherein the molecular weight as a free base of the lipopolymer is about 3.5 kD.   A nucleic acid delivery system comprising at least about 0.5 mg / ml filler excipient and nucleic acid, wherein at least a portion of the nucleic acid forms a complex and is condensed with a cationic lipopolymer suspended in an aqueous solvent. The cationic lipopolymer comprises a cationic polymer backbone independently covalently bonded to cholesterol and polyethylene glycol groups, wherein the molar ratio of cholesterol to cationic polymer backbone is from about 0.1 to about 10; A nucleic acid delivery system wherein the molar ratio of the functional polymer backbone is from about 0.1 to about 10.   36. The cationic polymer backbone is polyethyleneimine (PEI) and the molar ratio of PEG: PEI: cholesterol in the cationic lipopolymer is in the range of about 2-3: 1: 0.25-1 A nucleic acid delivery system as described.   37. The nucleic acid delivery system of claim 36, wherein the cationic lipopolymer has a free base molecular weight of about 3-4 kD.   38. The delivery system of claim 37, wherein the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 10: 1 to about 100: 1.   38. The delivery system of claim 37, wherein the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 11: 1 to about 20: 1.   24. The composition of claim 21, wherein the PEG: PEI: cholesterol molar ratio in the cationic lipopolymer is in the range of about 2-3: 1: 0.25-1.   An aqueous composition comprising a nucleic acid and a cationic lipopolymer that can be dried and reconstituted to a nucleic acid concentration of at least 0.5 mg / ml without nucleic acid aggregation.   42. The composition of claim 41, wherein the mixture of nucleic acid and cationic lipopolymer forms a complex.   43. The composition of claim 42, comprising a condensed nucleic acid.   38. The composition of claim 37, wherein the aqueous solution can be reconstituted into an isotonic solution.   40. The composition of claim 38, wherein at least about 30% by weight of the nucleic acid is condensed.   40. The composition of claim 39, wherein the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 10: 1 to about 100: 1.   41. The composition of claim 40, wherein the ratio of amine nitrogen in the cationic polymer backbone to phosphate in the nucleic acid is from about 11: 1 to about 20: 1.   42. The composition of claim 41, wherein the molar ratio of PEG: PEI: cholesterol in the cationic lipopolymer is in the range of about 2-3: 1: 0.25-1.   27. The dry composition of claim 26, wherein the composition can be reconstituted to a nucleic acid concentration of at least 0.5 mg / ml without nucleic acid aggregation.   42. The aqueous composition of claim 41 further comprising a filler excipient.   A method for stabilizing a nucleic acid delivery system comprising a nucleic acid and a cationic carrier, comprising a cationic lipopolymer comprising a cationic polymer backbone independently covalently bonded to cholesterol and polyethylene glycol groups and a filler excipient. Contacting the delivery system, wherein the molar ratio of cholesterol to cationic polymer backbone is from about 0.1 to about 10 and the molar ratio of polyethylene glycol to cationic polymer backbone is from about 0.1 to about 10 .   Cationic carriers are polyethyleneimine, poly (trimethyleneimine), poly (tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly (2-dimethylamino) ethyl Methacrylate, poly (lysine), poly (histidine), poly (arginine), cationized gelatin, dendrimer, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N- [1- (2,3 -Dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), 1- [2- (oleoyloxy) ethyl] -2-oleyl-3- (2-hydroxyethyl) imidazolinium Chloride (DOT M), 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3β- [N- (N ′, N '-Dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-cholesterol HCl) diheptadecylamide glycylspermidine (DOGS), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- ( 1,2-Dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N, N-dioleyl-N, N-dimethylammonium chloride (DODAC) and their 52. The method of claim 51, selected from the group consisting of combinations.

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